Abstract 3104: The mechanism of action of ADCs are dependent on Rab GTPases

Abstract Antibody-drug conjugates (ADCs) are therapeutic compounds designed to exert targeted toxicity as compared to conventional chemotherapy. The conjugation of a cytotoxic payload to an antibody trough a chemical linker serves to lower the systemic toxicity and thereby increase the therapeutic window. However, an enhanced understanding of the mechanism of action of ADCs is needed, as little is known about how these drugs are trafficked and processed within the cell. The tumor-targeted activity of ADCs depend on the presence of target molecules on the tumor cell surfaces, and the overall response of ADCs is generally dependent on the expression level of these surface receptors. However, ADCs are dependent on cellular uptake and intracellular trafficking for the cytotoxic payload to be released and reach its target within the cell. This process of endocytosis and subsequent intracellular trafficking is regulated by a group of small molecular switches called Rab GTPases. These proteins acts as key regulators of the endo/lysosomal machinery, and we hypothesized them to impact ADC efficacy. We used cell lines from breast and lung cancer origin to study the mechanism of action of different ADCs dependent on RAB5 and RAB4 expression. We have found that the makeup of the ADC - comprising the antibody moiety, linker design, and choice of payload - dictates how it is taken up and processed, and thereby determines which Rab GTPases are critical for its therapeutic effect. Our results demonstrate how ADC design influences intracellular trafficking and processing, and points towards linker design as a major regulator of the intracellular fate of an ADC. Citation Format: Astrid Medhus, Kay Oliver Schink, Ane Sager Longva, Olav Engebraaten, Kristian Berg, Anette Weyergang. The mechanism of action of ADCs are dependent on Rab GTPases. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3104.